Figure 28: MTcanA_rev sequence for cloning attempt #1. Sequence results were abnormally shorter than gene length. Sequence also contains a large amount of unknown nucleotide bases. Query was
Figure 29: MTcanA_for sequence for cloning attempt #1. Sequence results were abnormally shorter than gene length. Sequence also contains a large amount of unknown nucleotide bases.
When submitting a nucleotide BLAST on MTcanA for cloning attempt #1, a message of "No significant similarity" resulted.
Figure 30: MTcanA_rev sequence for cloning attempt #2. Sequence results looked better than the first attempt, however, the identity was only 93% and the query was 61%.
Figure 31: MTcanA_for sequence for cloning attempt #2. Sequence results looked better than the first attempt, however, the identity was only 93% and the query was 61%.
Oligo mix was remade from original primers. Tail primers, MTcanA_for and MTcanA_rev were also remade to rule out any possibilities for primer dimers. This time, secondary PCR showed much less primer dimers, though they still appeared. PCR squared will be performed to see if gel extraction will be possible.
Figure 24: MTcanA secondary PCR run at 58°C after remaking oligo mix and tail primers. First lane is 1kb ladder, second lane is primary PCR, third lane is secondary PCR. Some primer dimers are still visible, but are much more faint than before.
Figure 25: PCR of MTcanA run at 58.8°C. First lane is 1kb ladder, second lane is secondary PCR of MTcanA gene, and third lane is PCR squared of MTcanA gene.
Figure 26: Gel of cut pNIC-bsa4. First lane is 1 kb ladder. Second and third lane are Katherine's. Fourth lane is 30.9 ng/uL cut pNIC-bsa4, and fourth lane is 87.8 ng/uL cut pNIC-bsa4.
Virtual work has been started on the 1YLK protein with positive and negative control ligands. So far, only the control library of compounds has been concatenated.
Week 7 & 8
Secondary PCR was performed on a temperature gradient from 58°C to 68°C. Primer dimers still formed within all samples.
Figure 22: MTcanA secondary PCR temperature gradient. Lane 1 is 1kb ladder, lane 2 is 68°C, lane 3 is 67.3°C, lane 4 is 66.1°C, lane 5 is 64.3°C, lane 6 is 62°C, lane 7 is 60.3°C, lane 8 is 59°C, and lane 9 is 58°C. All samples show primer dimers as well as the MTcanA gene.
Secondary PCR was then done using 0.5uL of forward and reverse primers instead of 1.0uL. A lower annealing temperature of 56°C and the original annealing temperature of 58.8°C were used.
Figure 23: MTcanA secondary PCR. First lane is 1kb ladder. Second lane is 58.8°C sample. Third lane is 56°C sample. Both samples were made with 0.5uL of forward and reverse primers instead of the original 1.0uL. The second lane sample shows primer dimers. The third lane sample did not work, showing that 56°C is too low of an annealing temperature.
Week 5 & 6
Figure 20: Gel run for gel extraction on PCR sqaured for MTcanA. First lane is 1kb ladder. All other lanes are PCR squared samples. PCR squared samples show bands of contamination. These samples were used for gel extraction.
Gel extraction and PCR cleanup were performed using the PCR squared gels. Insufficient amount of MTcanA was retrieved due to contamination of the sample.
Figure 21: Secondary PCR of MTcanA with extended annealing temperature. First lane is 1kb ladder. Second lane is secondary PCR. No contamination is shown. Extra bands represent primer dimers as a result of incorrect annealing temperature.
Secondary PCR was repeated using extended annealing times. There was no contamination, however, the gel indicates that primer dimers have been created the secondary PCR sample. To solve this issue, a temperature gradient will be used for the annealing temperatures.
Week 3 & 4
pNICbsa4 from week 2 was nanodropped. The concentration was found to be 69.8 ng/uL. This sample of pNICbsa4 was submitted for DNA sequencing with the forward and reverse primers pLIC_for and pLIC_rev.
Figure 17: pNIC-bsa4 nanodrop (run 1) showing a concentration of 69.2 ng/uL. This was averaged with the concentration found in run 2 when submitting to DNA sequencing for an average concentration of 69.2 ng/uL.
Figure 18: pNIC-bsa4 nanodrop (run 2) showing a concentration of 70.4 ng/uL. This was averaged with the concentration found in run 1 when submitting to DNA sequencing for an averaged concentration of 69.8 ng/uL.
Primary and secondary PCR on MTcanA were completed and run on a gel. Secondary PCR shows two bands. Band at 516bp is the needed MTcanA. Lower band is a contaminant. PCR squared will be done to amplify MTcanA, however, gel extraction may need to be done if contaminant still remains after PCR squared.
Figure 19: Secondary PCR of MTcanA with new tail primers MTcanA_for and MTcanA_rev. First lane is 100bp ladder, second lane is 1kb ladder, third lane is primary PCR and fourth lane is seconday PCR showing two bands. Band at 516bp is MTcanA. Other band is contaminant.
Week 1 & 2**
New tail primers were ordered (MTcanA_for and MTcanA_rev) for target. These primers were each diluted from the original amount of 33.2 nMoles (MTcanA_for) and 33.4 nMoles (MTcanA_rev) to a stock dilution of 100uM. From this stock dilution, working dilutions of 20uM were made of each. An oligo mix for MTcanA was also made.
pNICbsa4 was grown up overnight and midiprepped.
Summer 2013
Week 8
Figure 14: Second attempt at secondary PCR of canA. First lane is 1kb ladder, second lane is seconday PCR. Procedure was not changed from the first attempt.
Figure 15: Third attempt at secondary PCR of canA. First lane is 1kb ladder, second lane is seconday PCR. Annealing temperature was changed to 58.8 degrees Celsius.
Figure 16: Fourth attempt at secondary PCR of canA. First lane is 1kb ladder, second lane is primary PCR, third lane is seconday PCR. Primary PCR was redone to ensure a good base for secondary PCR.
New tail primers were ordered and these will be used to re-run secondary PCR.
Week 7
Figure 13: Primary and Secondary PCR of canA. First lane is 1kb ladder, second lane is primary PCR, and third lane is seconday PCR. Secondary PCR was unsuccessful.
Fig 12: Restriction Enzyme digest gel; Lane 1: 1 kb ladder; Lane 2: uncut plasmid; Lane 3: EcoRI; Lane 4: PvuII; Lane 5: EcoRI and PvuII.
Week 5
PCR on pGBR22 was successful.
Figure 11: Fourth attempt (success!) at agarose gel of pGBR22 plasmid. Lane 1: 1kb ladder; Lane 2: sample A; Lane 3: sample B; Lane 4: sample C; Lane 5: sample D (control lane).
Figure 10: Third attempt at agarose gel of pGBR22 plasmid. Right lanes are Nicolet's and left lanes are Katherine's. Lane 1: ladder; Lane 2: sample A; Lane 3: sample B; Lane 4: sample C; Lane 5: sample D. No visible bands.
Week 3
PCR was done using pGBR22 from Week 2 and run on a gel for better results. This time it showed contamination.
Figure 9: First attempt at agarose gel of pGBR22 plasmid. Left lanes are Nicolet's and right lanes are Katherine's. Lane 1: ladder; Lane 2: sample A; Lane 3: sample B; Lane 4: sample C; Lane 5: sample D. Left lanes show contamination. Right lanes do not show results.
BL21(DE3) cells were used to transform an overnight culture of pNIC-Bsa4 in 100 ml LB. The next morning, the culture was transferred to a 500mL LB flask. 20 ml of the culture was transferred to a larger culture, stored in the shaking incubator, and checked every 30 minutes until an OD 600 of 0.625 was reached. This culture was then stored in the shaking incubator for 4 hours. The pNIC-Bsa4 culture was spun down and re-suspended in buffer to be stored at -80 degrees Celsius.
Week 2
Fig 6: Absorbance reading of sample 1 of pGBR22 plasmid.
Fig 7: Absorbance reading of sample 2 of pGBR22 plasmid.
Figure 8: First attempt at agarose gel of pGBR22 plasmid. Left lanes are Nicolet's and right lanes are Brenden's. Lane 1: ladder; Lane 2: sample A; Lane 3: sample B; Lane 4: sample C; Lane 5: sample D. No visible bands.
Week 1
Figure 1: E. coli cells transformed with 1 ng pGBR22 plasmid grown on LB+Amp Agar plates.
Figure 2: E. coli cells transformed with 5 ng pGBR22 plasmid grown on LB+Amp Agar plates.
Figure 3: E. coli cells transformed with 25 ng pGBR22 plasmid grown on LB+Amp Agar plates.
Transformation Efficiency:
Plate A, Fig. 1 (1 ng pGBR22): 3 Colonies
Plate B, Fig. 2 (5 ng pGBR22): 252 Colonies
Plate C, Fig. 3 (25 ng pGBR22): 1,920 Colonies
Fall 2013
Week 13 & 14Week 11 & 12
Missing analysis. more data? -UM
When submitting a nucleotide BLAST on MTcanA for cloning attempt #1, a message of "No significant similarity" resulted.
Week 9 & 10
Oligo mix was remade from original primers. Tail primers, MTcanA_for and MTcanA_rev were also remade to rule out any possibilities for primer dimers. This time, secondary PCR showed much less primer dimers, though they still appeared. PCR squared will be performed to see if gel extraction will be possible.
Virtual work has been started on the 1YLK protein with positive and negative control ligands. So far, only the control library of compounds has been concatenated.
Week 7 & 8
Secondary PCR was performed on a temperature gradient from 58°C to 68°C. Primer dimers still formed within all samples.
Secondary PCR was then done using 0.5uL of forward and reverse primers instead of 1.0uL. A lower annealing temperature of 56°C and the original annealing temperature of 58.8°C were used.
Week 5 & 6
Gel extraction and PCR cleanup were performed using the PCR squared gels. Insufficient amount of MTcanA was retrieved due to contamination of the sample.
Secondary PCR was repeated using extended annealing times. There was no contamination, however, the gel indicates that primer dimers have been created the secondary PCR sample. To solve this issue, a temperature gradient will be used for the annealing temperatures.
Week 3 & 4
pNICbsa4 from week 2 was nanodropped. The concentration was found to be 69.8 ng/uL. This sample of pNICbsa4 was submitted for DNA sequencing with the forward and reverse primers pLIC_for and pLIC_rev.
Primary and secondary PCR on MTcanA were completed and run on a gel. Secondary PCR shows two bands. Band at 516bp is the needed MTcanA. Lower band is a contaminant. PCR squared will be done to amplify MTcanA, however, gel extraction may need to be done if contaminant still remains after PCR squared.
Week 1 & 2**
New tail primers were ordered (MTcanA_for and MTcanA_rev) for target. These primers were each diluted from the original amount of 33.2 nMoles (MTcanA_for) and 33.4 nMoles (MTcanA_rev) to a stock dilution of 100uM. From this stock dilution, working dilutions of 20uM were made of each. An oligo mix for MTcanA was also made.
pNICbsa4 was grown up overnight and midiprepped.
Summer 2013
Week 8
New tail primers were ordered and these will be used to re-run secondary PCR.
Week 7
Updated target page: β-carbonic anhydrases
Week 6
Week 5
PCR on pGBR22 was successful.
Target Assigned: Beta-carbonic anhydrase (canA) (Mycobacterium tuberculosis)
Primers ordered: mtcanARev1284_FOR, mtcanARev1284_REV
Week 4
Week 3
PCR was done using pGBR22 from Week 2 and run on a gel for better results. This time it showed contamination.
BL21(DE3) cells were used to transform an overnight culture of pNIC-Bsa4 in 100 ml LB. The next morning, the culture was transferred to a 500mL LB flask. 20 ml of the culture was transferred to a larger culture, stored in the shaking incubator, and checked every 30 minutes until an OD 600 of 0.625 was reached. This culture was then stored in the shaking incubator for 4 hours. The pNIC-Bsa4 culture was spun down and re-suspended in buffer to be stored at -80 degrees Celsius.
Week 2
Week 1
Transformation Efficiency:
Plate A, Fig. 1 (1 ng pGBR22): 3 Colonies
Plate B, Fig. 2 (5 ng pGBR22): 252 Colonies
Plate C, Fig. 3 (25 ng pGBR22): 1,920 Colonies
ATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACAT
CCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAA
TAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGT
GTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGT
AGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCC
CATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTA
TTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCC
CCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGTAG
GTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCGAC
CATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATG
GTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG
CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTNNNAAANNTGTCNN
GNCAGCTGCATTAATGATCGGCNANNNNCGGGGNNANGNNNNTNGCGTNTGGGNNCTCTTCNNNTCCTCNNTCANNGACT
CNNNNNNNTCNNNCNNNNNNNNNNNNNNNNNGNNTCANNNNNNNNNANGNNNGNNNNNNNNNNNNNNNNNNNNNNNNNNN
ANNNNNNNGNAANANNNNNNNNNNNNNNNNNNNNAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNN
NNN